Monoclonal antibodies (Mabs) have become the most successful protein drugs being used for the treatment of a variety of diseases, including cancers, transplantation, viral infection, etc. However, the concept of magic bullet took more than 25 years to realize, because there were problems associated with the use of monoclonal antibodies. One of the main problems stems from the original source of most monoclonal antibodies, which are of rodent and murine origin. Repeated injections of these foreign proteins into human would inevitably result in the elicitation of host immune responses against the antibodies: the so-called human anti-mouse antibody (HAMA) responses. Although earlier attempts to use the techniques of molecular engineering to construct chimeric antibodies (for example, mouse variable regions joined to human constant regions) were somewhat effective in mitigating HAMA responses, there remains a large stretch of murine variable sequences constituting ⅓ of the total antibody sequence that could be sufficiently immunogenic in eliciting human anti-chimeric antibody (HACA) responses. A more advanced improvement in antibody engineering has recently been utilized to generate humanized antibodies in which the complementarity determining regions (CDR's) from a donor mouse or rat immunoglobulin are grafted onto human framework regions (for example, EPO Publication No. 0239400, incorporated herein by reference). The process is called “humanization”, or “CDR-grafting”. The original concept of humanization describes the direct grafting of CDR's onto human frameworks, reducing the non-human sequences to less than 5%, and thereby the HAMA and HACA responses. However, direct replacement of framework sequences without further modifications can result in the loss of affinity for the antigen, sometimes as much as 10-fold or more (Jones et al., Nature, 321:522-525, 1986; Verhoyen et al., Science, 239:1534-1536, 1988). To maintain the affinity of the CDR-grafted or humanized antibody, substitutions of a human framework amino acid of the acceptor immunoglobulin with the corresponding amino acid from a donor immunoglobulin at selected positions will be required. The positions where the substitution takes place are determined by a set of published criteria (U.S. Pat. No. 5,85,089; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,693,761; incorporated herein by reference). However, the presence of murine amino acids within stretches of human framework sequences can be immunogenic in the generation of new T- and B-cell epitopes. Moreover, the identification of the proper framework amino acids to be replaced can sometimes be difficult, further reducing the chances of success in humanization without significant impacts on the specificity and affinity of the humanized antibody.
New and improved means for producing re-engineered immunoglobulin with reduced or eliminated immunogenicity while maintaining the specificity and affinity of the parent antibody are therefore needed. Preferably, the re-engineered immunoglobulin should contain no FR amino acid substitutions from the parent antibody, which can be a likely source of immunogenic epitopes for T- or B-cells. However, the approach also offers flexibility in the sequence design where few murine residues or a stretch of murine sequences can be included in the final design, with the ultimate goal of reducing immunogenicity while maintaining specificity and affinity of the resultant antibody for human uses. The present invention describes the methods and approaches in fulfilling these goals.